Yutaka Nakaya

Dietary Phosphorus Acutely Impairs Endothelial Function

Excessive dietary phosphorus may increase cardiovascular risk in healthy individuals as well as in patients with chronic kidney disease, but the mechanisms underlying this risk are not completely understood. To determine whether postprandial hyperphosphatemia may promote endothelial dysfunction, we investigated the acute effect of phosphorus loading on endothelial function in vitro and in vivo. Exposing bovine aortic endothelial cells to a phosphorus load increased production of reactive oxygen species, which depended on phosphorus influx via sodium-dependent phosphate transporters, and decreased nitric oxide production via inhibitory phosphorylation of endothelial nitric oxide synthase. Phosphorus loading inhibited endothelium-dependent vasodilation of rat aortic rings. In 11 healthy men, we alternately served meals containing 400 mg or 1200 mg of phosphorus in a double-blind crossover study and measured flow-mediated dilation of the brachial artery before and 2 h after the meals. The high dietary phosphorus load increased serum phosphorus at 2 h and significantly decreased flow-mediated dilation. Flow-mediated dilation correlated inversely with serum phosphorus. Taken together, these findings suggest that endothelial dysfunction mediated by acute postprandial hyperphosphatemia may contribute to the relationship between serum phosphorus level and the risk for cardiovascular morbidity and mortality.

Coronary Atherosclerosis Is Associated With Macrophage Polarization in Epicardial Adipose Tissue

OBJECTIVES The purpose of this report was to assess the link between macrophage polarization in epicardial adipose tissue and atherosclerosis in patients with coronary artery disease (CAD). BACKGROUND Macrophage accumulation enhances chronic inflammation in adipose tissue, but macrophage phenotypic change in human epicardial adipose tissue and its role in atherogenesis are unknown. METHODS Samples were obtained from epicardial and subcutaneous adipose tissue during elective cardiac surgery (CAD, n = 38; non-CAD, n = 40). Infiltration of M1/M2 macrophages was investigated by immunohistochemical staining with antibodies against CD11c and CD206, respectively. Expression of pro- and anti-inflammatory adipocytokines in adipose tissue was evaluated by real-time quantitative polymerase chain reaction. RESULTS Infiltration of macrophages and expression of pro- and anti-inflammatory cytokines were enhanced in epicardial fat of patients with CAD compared with that in non-CAD patients (p < 0.05). The ratio of M1/M2 macrophages was positively correlated with the severity of CAD (r = 0.312, p = 0.039). Furthermore, the expression of pro-inflammatory cytokines was positively correlated, and the expression of anti-inflammatory cytokines was negatively correlated with the ratio of M1/M2 macrophages in epicardial adipose tissue of CAD patients. By contrast, there was no significant difference in macrophage infiltration and cytokine expression in subcutaneous adipose tissue between the CAD and non-CAD groups. CONCLUSIONS The ratio of M1/M2 macrophages in epicardial adipose tissue of CAD patients is changed compared with that in non-CAD patients. Human coronary atherosclerosis is associated with macrophage polarization in epicardial adipose tissue.

Decreased food intake and body weight in pancreatic polypeptide-overexpressing mice

BACKGROUND & AIMS Pancreatic polypeptide (PP) is a 36-amino acid hormone produced by F cells within the pancreatic islets and the exocrine pancreas. The definitive function of PP in mammalian physiology remains to be determined. This study examined the effects of chronic overexpression of PP through the development of PP transgenic mice. METHODS PP transgenic mice were created by using mouse PP complementary DNA under the control of the cytomegalovirus immediate early enhancer-chicken beta-actin hybrid promoter (pCAGGS expression vector). RESULTS A unique line of transgenic mice was created that overexpresses PP in the pancreatic islets with low levels of expression in other tissues including the brain. Plasma PP concentrations were more than 20 times higher than those of control littermates. However, PP overproduction led to postnatal lethality in half of the pups because of markedly decreased milk intake. The remaining PP transgenic mice gained less weight with specifically reduced food intake and fat mass compared with controls, a result that was more evident in male than in female mice. The transgenic mice exhibited a reduced rate of gastric emptying of a solid meal but had normal oxygen consumption and fasting leptin levels. Immunoneutralization with anti-PP antiserum reversed the phenotypic changes of transgenic animals. CONCLUSIONS PP could be involved in feeding and body weight regulation partly through regulation of gastric emptying.

Atrial natriuretic factor and isosorbide dinitrate modulate the gating of ATP-sensitive K+ channels in cultured vascular smooth muscle cells.

The effects of atrial natriuretic factor (ANF) and isosorbide dinitrate (ISDN), activators of particulate and soluble guanylate cyclase, respectively, on K+ currents were investigated in patch-clamp recordings of smooth muscle cells cultured from rat thoracic aorta. In the cell-attached patch configuration, ANF enhanced Ca(2+)-activated K+ (KCa) channel activities as reported previously. When KCa channels were blocked with 1 mmol/L tetraethylammonium or 10(-7) mol/L charybdotoxin, ANF and ISDN applied to the bathing solution activated ATP-sensitive K+ (KATP) channels without altering channel conductance. Pretreatment with methylene blue, a guanylate cyclase inhibitor, abolished the effects of ISDN on KATP channels, whereas 8-bromo-cGMP activated these channels, suggesting that the effects of ISDN on KATP channels were mediated by cGMP. Our results suggest that vasorelaxant agents that increase intracellular cGMP concentrations modulate the gating of two major potassium channels, ATP sensitive and Ca2+ activated, that might play an important role in controlling vascular tone by changing the membrane potential.

Nonendothelial-derived nitric oxide activates the ATP-sensitive K+ channel of vascular smooth muscle cells

To determine whether endogenous nitric oxide (NO) opens the ATP‐sensitive K+ channel (KATP channel), we investigated the effect of nonendothelial‐derived NO on this channel in cultured smooth muscle cells of the porcine coronary artery by the patch‐clamp technique. In the cells pretreated with endotoxin, the addition of 10−4 M l‐arginine generated NO and activated the KATP channel. Activation of this channel was suppressed by pretreatment with 10−3 M N G‐methyl‐l‐arginine or 10−3 M N x‐nitro‐l‐arginine methyl ester, each of which is a specific antagonist of the l‐arginine‐NO pathway, and by 10−6 M Methylene blue, which blocks guanylate cyclase. The activation of the KATP channel by l‐arginine‐NO pathway is expected to produce hyperpolarization of the cell membrane and relaxation of vascular smooth muscle cells.

Effect of Exercise Training and Food Restriction on Endothelium-Dependent Relaxation in the Otsuka Long-Evans Tokushima Fatty Rat, a Model of Spontaneous NIDDM

We investigated whether endothelial function may be impaired in the Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of spontaneous NIDDM. The effect of exercise training and food restriction on endothelial function was also studied. OLETF rats were divided into three groups at age 16 weeks: sedentary, exercise trained, and food restricted (70% of the food intake of sedentary rats). Otsuka Long-Evans Tokushima rats were used as the age-matched nondiabetic controls. Endothelium-dependent relaxation of the thoracic aorta induced by histamine was significantly attenuated in the sedentary or food-restricted rats, and exercise training improved endothelial function. Relaxation induced by sodium nitroprusside, a donor of nitric oxide, did not differ significantly among groups. Both exercise training and food restriction significantly suppressed plasma levels of glucose and insulin and serum levels of triacylglycerol and cholesterol and reduced the accumulation of abdominal fat. Insulin sensitivity, as measured by the hyperinsulinemic- euglycemic clamp technique, was significantly decreased in sedentary rats but was enhanced in exercise- trained and food-restricted rats. The urinary excretion of nitrite was significantly decreased in sedentary and food-restricted rats compared with nondiabetic rats and was significantly increased in exercise-trained rats. These results indicate that exercise training, but not food restriction, prevents endothelial dysfunction in NIDDM rats, presumably due to the exercise-induced increase in the production of nitric oxide.

Hydrogen peroxide-induced vascular relaxation in porcine coronary arteries is mediated by Ca2+-activated K+ channels

SummaryHydrogen peroxide (H2O2) elicited concentration-dependent relaxation of endothelium-denuded rings of porcine coronary arteries. The relaxation induced by the H2O2 was markedly attenuated by 10μM 1H-[1,2,4]oxadiazolo [4,3,a]quinoxalin-1-one (ODQ), an inhibitor of soluble guanylate cyclase, or by 100nM charybdotoxin, an inhibitor of large-conductance Ca2+-activated K+ (KCa) channels. A combination of the ODQ and charybdotoxin abolished the H2O2-induced relaxation. Pretreatment with 25 μM of an Rp stereoisomer of adenosine-3′,5′-cyclic monophosphothioate (Rp-cAMPS), 20μM glibenclamide, or 1mM 4-aminopyridine did not affect the vascular response to H2O2. The presence of catalase at 1000U/ml significantly attenuated the H2O2-induced relaxation. Exposure of cultured smooth muscle cells to H2O2 activated KCa channels in a concentration-dependent manner in cell-attached patches. Pretreatment with catalase significantly inhibited the activation of KCa channels. Rp-cAMPS did not inhibit the H2O2-induced activation of KCa channels. The activation of KCa channels by H2O2 was markedly decreased in the presence of ODQ. However, even in the presence of ODQ, H2O2 activated KCa channels in a concentration-dependent manner. In inside-out patches, H2O2 significantly activated KCa channels through a process independent of cyclic guanosine 3′,5′-monophosphate (cGMP). In conclusion, H2O2 elicits vascular relaxation due to activation of KCa channels, which is mediated partly by a direct action on the channel and partly by activation of soluble guanylate cyclase, resulting in the generation of cGMP.

Exendin-4, a GLP-1 receptor agonist, directly induces adiponectin expression through protein kinase A pathway and prevents inflammatory adipokine expression

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that has been used as a drug injected subcutaneously for treatment of type 2 diabetes. Many studies have revealed molecular targets of Ex-4, but its influence on adipokines has not been determined. Our study showed that Ex-4 induced secretion of adiponectin into the culture medium of 3T3-L1 adipocytes. This effect of Ex-4 is due to increased adiponectin mRNA level through the GLP-1R. Both forskolin and 3-isobutyl-1-methylxanthine (IBMX), which may finally elevate cyclic adenosine monophosphate (cAMP) concentration, prevented the induction of adiponectin expression by Ex-4. Moreover, H89, a protein kinase A inhibitor, blocked the effect of Ex-4 on adiponectin. On the other hand, Ex-4 decreased the mRNA levels of inflammatory adipokines. The results indicate that Ex-4 directly promotes adiponectin secretion via the protein kinase A pathway in 3T3-L1 adipocytes and may ameliorate insulin resistance.

Mechanism of activation of the Ca2+-activated K+ channel by cyclic AMP in cultured porcine coronary artery smooth muscle cells

Abstract Activation of the Ca 2+ -activated K + channel (K Ca -channel) by adenosine 3′, 5′-cyclic monophosphate (cAMP) and cAMP-dependent protein kinase (A-kinase) in cultured smooth muscle cells from porcine coronary artery was investigated using the patch-clamp technique. In cell-attached patches, the K Ca -channel was activated when forskolin (10 μM) was applied to the bath. In excised inside-out patches, application of 50 μM cAMP to the bath activated the K Ca -channel in the presence of A-kinase (10 units/ml) and ATP (1 mM). In addition, the K Ca -channel was activated directly by application of cAMP to the cytoplasmic side of the membrane in the absence of A-kinase. The activation by cAMP or by A-kinase required intracellular Ca 2+ , and was enhanced by increase of intracellular Ca 2+ . At a low concentration (3×10 −7 M) of Ca 2+ , more than 2 mM cAMP was required for activation of the K Ca -channel, but with 10 −6 M Ca 2+ , 100 μM cAMP was sufficient for activation. These results suggest that there are two mechanisms of activation of the K Ca -channel by cAMP : direct activation, and indirect activation via phosphorylation of the channel by A-kinase.

New water disinfection system using UVA light-emitting diodes

Aim:  To evaluate the ability of high‐energy ultraviolet A (UVA) light‐emitting diode (LED) to inactivate bacteria in water and investigate the inactivating mechanism of UVA irradiation.